Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados / Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.

Influence of osteopontin in bovine uterine tube fluid on sperm binding and fertilization in RCA-1 lectin-treated oocytes.

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 microl of fertilization medium (FM); (ii) 250 microl of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 microl of FM with 250 microl of NLAUTF and 4 microl of RCA-1 lectin; (iv) 250 microl of FM with 250 microl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 microl of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 microg heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.

Oviductal fluid proteins associated with the bovine zona pellucida and the effect on in vitro sperm-egg binding, fertilization and embryo development.

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding.

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.

Pre-treatment of cattle semen or oocytes with purified milk osteopontin affects in vitro fertilization and embryo development.

This study was designed to investigate the effects of pre-incubating cattle spermatozoa or matured oocytes with purified osteopontin (OPN) from cattle milk on fertilization in cattle and embryonic development in vitro. There were two different experiments, semen from six mature Holstein bulls (Bos Taurus) was frozen with different concentrations of OPN (0, 1, 10, 100 microg/mL). Matured cattle oocytes were also pre-treated with OPN (0, 10, 100 microg/mL). In both experiments, pre-treated oocytes or frozen semen, was processed for in vitro fertilization and embryo development. Significantly more oocytes were fertilized when using frozen semen with 10 microg/mL OPN (bull 2=85+/-4% and bull 5=78+/-4%) than without OPN (bull 2=75+/-4% and bull 5=69+/-4%). Those bulls also had increase in cleavage and embryo development (bull 2=85+/-3%, 41+/-1.9%; bull 5=76+/-2%, 37+/-1.8%) compared with control (bull 2=75+/-3%, 30+/-2%; bull 5=68+/-2%, 29+/-2%). Incubating matured oocytes in 10 microg/mL OPN (87+/-3%) and 100 microg/mL OPN (88+/-3%) significantly increased fertilization than control (73+/-3%). OPN also improve cleavage, and embryo development in treatments with 10 microg/mL OPN (82.7+/-1.3%; 31.7+/-1.4%) and 100 microg/mL OPN (85.8+/-1.3%; 33.8+/-1.5%) when compared with control (74.1+/-1.3%; 24.2+/-1.2%). These data suggest that both, spermatozoa from some bulls and oocytes may associate with OPN, suggesting a facilitory role on in vitro fertilization and embryo development.

Influence of arginine-glycine-aspartic acid (RGD), integrins (alphaV and alpha5) and osteopontin on bovine sperm-egg binding, and fertilization in vitro.

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<0.05) fertilization compared to the in vitro-fertilized control. These data provided evidence that integrin ligands existed on bovine oocytes and spermatozoa that contained RGD recognition sequences, and that antibody to OPN, a protein that contains that RGD sequence, was capable of reducing sperm-egg binding and fertilization in vitro.

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine.

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.

Expression pattern of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) system members in bovine corpus luteum endothelial cells during treatment with FGF-2, VEGF or oestradiol.

The development of the corpus luteum (CL) is accompanied by very active angiogenesis. We hypothesize that during this process endothelial cells (EC) are under the control of several angiogenic factors and steroids. The aim of this study was to examine the expression of the angiogenic growth factor systems - fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) - in EC derived from the bovine CL. Endothelial cells were cultured in serum-free medium and treated for 24 h with different concentrations of oestradiol (range from 10(-13) to 10(-5) mol/l), VEGF or FGF-2 (1, 10 and 100 ng/ml, respectively) and compared with untreated controls. Cells were harvested, total RNA extracted and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Treatment with oestradiol or FGF-2 stimulated the expression of FGF-2, but VEGF treatment showed no effect on the FGF-2 expression. FGF-2 or VEGF treatment resulted in an up-regulation of the FGF receptor (FGFR) mRNA. However, no FGF-1 expression was detected in EC. For the VEGF system, treatment with FGF-2, VEGF or oestradiol did not affect VEGF expression. However, the presence of FGF-2 in the medium up-regulated the expression of both VEGF receptors (VEGFR-1 and VEGFR-2), whereas oestradiol or VEGF treatment showed no effect on the expression of these receptors. Our results reveal that functional angiogenic growth factor systems were expressed in vitro in bovine EC derived from the CL. This suggests that the angiogenic FGF and VEGF system members were regulated by FGF or VEGF, but not by oestradiol-17beta.

Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs.

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES-1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles.

Evidence for the role of oviduct secretions in sperm function, fertilization and embryo development.

The oviduct is a dynamic organ which facilitates gamete function, fertilization and embryo development. Secretions of the oviduct, recovered by tissue culture or cannulation techniques have been used to define the composition of the oviduct milieu, as well as functions associated with stage of the reproductive cycle or region of the oviduct. Several oviduct proteins have been shown to associate with the gametes and embryos. Ongoing studies are directed at identifying oviduct proteins and determining their function. Oviduct-specific glycoproteins (OSG) have been purified from the oviduct and shown in vitro to have positive affects on sperm capacitation, sperm-ovum binding, ovum penetration and embryo development. Osteopontin, another oviduct secretion, also has been shown to stimulate fertilization and embryo development. The picture emerging is that some components of the oviduct milieu have overlapping functions to collectively provide a failsafe system to ensure fertility in vivo so that success is not dependent on a single component.

Induction of the acrosome reaction in bull spermatozoa with plasmin.

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well-documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 microg ml-1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg ml-1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.

Expression and presence of osteopontin and integrins in the bovine oviduct during the oestrous cycle.

Osteopontin and integrin alpha(v)beta(3) are known to mediate cell-cell attachment and cell migration. Western blot analysis was used to demonstrate the presence of osteopontin in oviductal fluid collected from ampullar and isthmic regions. Three different osteopontin isoforms of 55 kDa, 48 kDa and 25 kDa were detected in the oviductal fluid. Each isoform was observed during the luteal and non-luteal phases and in both ampullar and isthmic fluids. The 25 kDa osteopontin was the most prevalent isoform in oviductal fluid except in isthmic fluid during the non-luteal phase of the oestrous cycle. RT-PCR was performed with RNA from oviductal cells collected from cows in the post-ovulatory, early to mid-luteal, late luteal or pre-ovulatory stages of the oestrous cycle to reveal the oviduct as a site of osteopontin and integrin synthesis. Only one osteopontin mRNA transcript was detected, and amounts did not vary throughout the oestrous cycle. In contrast, the relative expression of the integrin subtypes alpha(v) and beta(1) during the late luteal phase was lower compared with the other oestrous cycle phases. Integrin beta(3) mRNA content increased significantly from the lowest level during the late luteal phase to the highest level before ovulation. In conclusion, differential presence of osteopontin isoforms and integrins in the bovine oviduct throughout the oestrous cycle indicate that osteopontin-integrin interactions have functional roles in normal oviduct physiology which may potentially influence interactions between the gametes, the embryo, and the epithelium.

Differential expression of extracellular matrix components in the bovine oviduct during the oestrous cycle.

Components of the extracellular matrix take part in tissue rebuilding as well as activating surface-bound growth factors. In the present study, expression and selected activities of urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), their inhibitors (plasminogen activator inhibitor 1 (PAI-1) and tissue inhibitors of metalloproteinases (TIMPs)) were examined in bovine oviducts by RT--PCR, ribonuclease protection assay and activity assays. A high content of mRNA encoding for uPA was detected before ovulation with a three-fold decrease after ovulation. In contrast, PAI-1 expression appeared to be stable during the oestrous cycle. Oviductal flushings produced caseinolytic zones in zymograms containing plasminogen at approximately 50 kDa and 28 kDa. An activity assay for uPA showed highest net activity during the early to mid-luteal phase. Increased TIMP-1 and MMP-2 mRNA concentrations were found around the time of ovulation compared with the luteal phase. In contrast, MMP-1 mRNA transcripts were enriched during the early to mid-luteal phase. Gelatin zymograms detected a 70--72 kDa protease activity showing an oestrous cycle-dependent activity with highest activity before ovulation. Reverse zymography detecting TIMPs revealed proteins between 21 kDa and 24 kDa. Only for the smallest (21 kDa) protein were amounts increased around the time of ovulation compared with the luteal phase. The observation that several extracellular matrix components were regulated distinctly in bovine oviducts indicates that local interactions between these components, growth factors, gametes and the embryo are possible and may influence fertilization and early embryonic development.

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.

Immunocontraception of white-tailed deer with GnRH vaccine.

PROBLEM: Reduction of excess numbers of white-tailed deer (Odocoileus virginianus) is an example of a potential use for immunocontraception as a means of wildlife population management. METHOD OF STUDY: A 4 year multifaceted study was conducted to determine the long term effects of gonadotropin releasing hormone (GnRH) contraceptive vaccine on the fertility and behavior of female and male white-tailed deer. Deer were monitored for breeding behavior, hormone levels, pregnancy, fawning and GnRH specific antibody levels. RESULTS: Treatment lead to reduced fawning rates, altered estrus behavior, reduced concentrations of progesterone, contraception and failure to maintain pregnancy following conception. GnRH immunized does bred to untreated bucks had an 88% reduction in fawning caused by either immunocontraception or immunocontragestion. The vaccine effect is reversible, directly related to the antibody titer. Infertility lasted up to two years without boosting. GnRH immunized bucks demonstrated no interest in sexual activity when paired with control females. Depending on the immunization schedule, antlers either dropped early or remained in velvet. CONCLUSIONS: The results of this study demonstrate that GnRH vaccine is effective in inducing a reversible infertility in white-tailed deer, the infertility lasting up to two years without boosting.

Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis.

Lipocalin-type prostaglandin D synthase in semen has been associated with male fertility, although this relationship is not well defined. To gain insight into potential mechanisms, the objective of the present study was to immunocytochemically localize lipocalin-type prostaglandin D synthase within the testis, efferent ducts, and 4 segments of mouse epididymis. In the testis, immunoperoxidase staining was localized within the Sertoli cells only at stages VI-VIII of the spermatogenic cycle, which is just prior to spermiation. Intense staining was also evident throughout the interstitial tissue, including Leydig cells. The entire epithelium of the efferent ducts, including ciliated and nonciliated cells, was immunoreactive. A distinct pattern of immunostaining for lipocalin-type prostaglandin D synthase was observed in different regions of epididymis, suggesting a possible role in sperm maturation. Staining for lipocalin-type prostaglandin D synthase was strikingly absent in the initial segment. In caput epididymidis, staining was evident throughout the cell cytoplasm of principal cells with some cells more intensely stained than adjacent ones. In the corpus region, overall staining intensity decreased and appeared to be concentrated in the apical region of principal cells, but some cells were completely unreactive. Reaction product in the cauda region was heavily concentrated on microvilli and within the epididymal lumen. In all epididymal regions, expression of lipocalin-type prostaglandin D synthase was specific to epithelial principal cells; no immunoreactivity was apparent in other cell types. The specific localization of lipocalin-type prostaglandin D synthase within the testicular interstitial tissue, Sertoli cells, and principal cells of caput epididymidis strongly suggests that this protein plays an integral role in both the development and maturation of sperm.

Expression of the lipocalin-type prostaglandin D synthase gene in the reproductive tracts of Holstein bulls.

The aim of this study was to localize expression of the prostaglandin D synthase gene in the reproductive tracts of Holstein bulls using northern blotting and in situ hybridization. For northern blotting, a digoxigenin-labelled prostaglandin D synthase cDNA probe was used to probe blots containing RNA isolated from the testes, epididymides, vas deferens, ampullae, seminal vesicles, prostate and bulbourethral glands of bulls. The digoxigenin-labelled cDNA for the bovine homologue of prostaglandin D synthase hybridized to a single band (approximately 0.9 kb) to RNA samples from the caput, corpus and cauda epididymides, as well as RNA samples from the vas deferens and the ampulla. The probe also detected a single band in testis samples, although the transcript size was slightly larger (approximately 1.0 kb) than the transcript found in the other tissues. The highest expression of prostaglandin D synthase was observed in the testes and caput epididymides. Prostaglandin D synthase transcripts were not found in the seminal vesicles or the prostate or bulbourethral glands using northern blotting. For in situ hybridization, antisense and sense riboprobes were synthesized and used to hybridize to cryosections obtained from the reproductive tissues of bulls. In situ hybridization of bull testes showed that prostaglandin D synthase transcripts were present within the germ cells in the adluminal compartment of the seminiferous tubules containing round and elongated spermatids, indicating that expression varied with stage of development of the seminiferous tubules. Prostaglandin D synthase expression was observed in the epithelial cells of the epididymides with greatest expression occurring in the caput epididymidis. Some expression was also observed in the epithelial cells of the vas deferens and a few cells of some lobules in the prostate and bulbourethral glands. Expression of the prostaglandin D synthase gene was not detected in ampullae or seminal vesicles by in situ hybridization.

Immunocontraception of white-tailed deer using native and recombinant zona pellucida vaccines.

We conducted a 2-year feasibility study with native porcine zona pellucida (PZP) vaccine and three recombinant rabbit zona pellucida vaccines (RC55, RC75a and a combination of RC55, RC75a and RC75b) as an initial phase of developing a recombinant immunocontraceptive vaccine to control reproduction in overpopulated herds of white-tailed deer (Odocoileus virginianus). Forty captive white-tailed does were divided into five groups (one sham and four treated), of eight each and injected with a 500microg prime dose of vaccine. Each prime dose was followed by a 300microg booster dose at 3-7 weeks post prime. The frequency and number of months of observed breeding were higher in PZP immunized does than in sham controls. Although the antibody titers of the three recombinant groups were 1000 or less, as compared with the PZP group with titers often over 128,000, the fawning rates of the two recombinants were significantly lower than that of the control group. The combined antigen group did not have a significantly lower fawning rate.

Osteopontin gene expression in the Holstein bull reproductive tract.

The objective of this study was to localize gene expression of osteopontin in the Holstein bull reproductive tract using Northern blot analysis and in situ hybridization. For Northern blot analysis, a digoxigenin-labeled osteopontin complementary deoxyribonucleic acid (cDNA) was used to probe blots containing total ribonucleic acid (RNA) isolated from the testis, epididymis, vas deferens, ampulla, seminal vesicle, prostate, and bulbourethral glands. The digoxigenin-labeled cDNA for the bovine homologue of osteopontin was hybridized to a single band at approximately 1.6 kb to RNA samples from the ampulla and seminal vesicle. For in situ hybridization studies, antisense and sense riboprobes were synthesized and used to hybridize cryosections that had been obtained from bull reproductive tissues. In situ hybridization of the bull testis detected osteopontin messenger RNA in the developing germ cells. Osteopontin gene expression was detected only in seminiferous tubules that contained elongated spermatids, which suggests that expression varies with the stage of the seminiferous epithelium. Within the epididymis, silver grains were distributed over the sperm that were located within the lumen of the caput, corpus, and cauda epididymis. Osteopontin expression was primarily observed in the epithelial cells of the ampulla. Antisense riboprobes also hybridized to sperm that were located within the lumen of the ampulla, confirming the presence of osteopontin transcripts in the haploid male gamete.

Effects of accessory sex gland fluid on viability, capacitation, and the acrosome reaction of cauda epididymal bull spermatozoa.

The effect of accessory sex gland fluid (AGF) on viability and acrosomal integrity of spermatozoa was examined with cauda epididymal spermatozoa and AGF from the same Holstein bull (n = 6). Surgical cannulation of the vasa deferentia enabled the separate collection of cauda epididymal effluent and AGF from each bull. Cauda epididymal effluent was incubated with either AGF collected from the same bull or medium alone. Following coincubation, spermatozoa (5 x 10(7) sperm/mL) were incubated in medium alone or under capacitating conditions (10 microg/mL heparin) for 16 hours. Every 2 hours, an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg/mL) to induce the acrosome reaction in capacitated spermatozoa. Sperm motility decreased over time regardless of treatment. Overall, spermatozoa incubated in AGF had fewer acrosome-intact live spermatozoa than did those not incubated in AGF. Viability was significantly (P < .05) compromised over time when spermatozoa were exposed to AGF, compared with those not preincubated in AGF. Significantly more (P < .05) acrosome-reacted live spermatozoa were seen following exposure to heparin and lysophosphatidylcholine when spermatozoa were not preincubated in AGF. We conclude that exposure of spermatozoa to AGF accelerates cell death and that rapid removal of spermatozoa from seminal plasma is critical for maximal viability.